Functional characterization of the antibiotic resistance reservoir in the human microflora

Abstract

To understand the process by which antibiotic resistance genes are acquired by human pathogens, we functionally characterized the resistance reservoir in the microbial flora of healthy individuals. Most of the resistance genes we identified using culture-independent sampling have not been previously identified and are evolutionarily distant from known resistance genes. By contrast, nearly half of the resistance genes we identified in cultured aerobic gut isolates (a small subset of the gut microbiome) are identical to resistance genes harbored by major pathogens. The immense diversity of resistance genes in the human microbiome could contribute to future emergence of antibiotic resistance in human pathogens.

Figure 1

Distributions of (A) nucleotide identities and (B) amino acid identities for 93 resistance genes identified from DNA extracted directly from saliva and fecal samples to the most similar resistance gene from any organism (white bars) as well as the most similar resistance gene harbored by a pathogenic isolate (black bars) in GenBank (Table S3)(24). Two resistance genes with no significant similarity to sequences in GenBank are not shown.

Figure 2

Antibiotic resistance profiles of cultured aerobic gut microbiome isolates. (A) Heat map displaying resistance profiles of 572 aerobic bacterial isolates obtained across 3 different sampling times from two human gut microbiomes. 7436 growth measurements of aerobic cultured microbiome isolates after 24 hrs at 37 °C in Luria Broth containing one of 13 antibiotics at concentrations between 20 and 100 mg/mL that prevent the growth of wild type E.coli (Table S1) are displayed as linear color-scaled bars. White denotes no growth, and color intensity is proportional to growth in the presence of antibiotic, scaled to growth in the absence of antibiotic per individual isolate. (B) Percentage of aerobic gut isolates resistant to each of 13 antibiotics. Each data point represents the mean number of isolates resistant to each antibiotic, and error bars represent the standard deviation of this mean value from each of the three sampling times. Histogram depicting the distribution of the number of different antibiotics that aerobic (C) gut microbiome 1 and (D) gut microbiome 2 isolates are resistant to.

Figure 3

Distributions of (A) nucleotide identities and (B) amino acid identities for 114 resistance genes identified from cultured aerobic gut isolates to the most similar resistance gene from any organism (white bars) as well as the most similar resistance gene harbored by a pathogenic isolate (black bars) in GenBank (Table S4)(24). One resistance gene with no significant similarity to sequences in GenBank is not shown.

Figure 4

The phylogenetic relationship of unique beta-lactamases derived from gut and oral microbiomes from healthy humans is displayed as an unrooted neighbor-joining tree (24). Except for nodes indicated, bootstrap values = 1000. Scale bar is in fixed amino acid substitutions per sequence position. Border colors of squares denote sequences derived from cultured aerobic gut isolates (blue), metagenomic DNA (red) or both (yellow). Internal shading of each square represents percentage amino acid identity to the most similar sequence in GenBank, with a linear gradient between 100% identity (white) and 35% identity (black). Sequence groups are labeled according to standard nomenclature (Table 1)(24, 32).